Neuronal cell death can occur through two principal mechanisms; necrosis and apoptosis. The molecular mechanisms involved in these two pathways for cell death, however, probably contain some overlap. Currently, it is believed that the major effectors of the apoptotic program are the ICE/CeD-3 family of cysteine proteases (caspases). Despite emerging evidence that ICE-family proteases play important roles as effectors of neuronal cell death, little is currently known about which types of neural cells express particular members of the multi-gene family under normal circumstances and how the expression and activation of these proteins might change during pathological situations. Moreover, it is controversial where some caspases are located within cells, given recent evidence that certain caspases may residue within mitochondria, become released into the cytosol following exposure to apoptotic stimuli. In addition, caspase-activators such as cytochrome C can be released from mitochondria during apoptosis but the relevance of this process to in vivo neuronal cell death during ischemia or other pathological situations has not been explored. The following specific aims are therefore proposed to explore the expression, activation, and function of ICE family proteases in animal models of neuronal cell death: (1) Antibodies will be generated, permitting immunohistochemical detection of several members of the ICE family in brain tissue sections; (2) The normal patterns of expression and intracellular locations of ICE-family proteases will be determined in the rat and mouse brains; (3) Changes in the expression, location, and processing of these proteins will be documented using rat or mouse models of transient global and focal ischemia; (4) An in vitro model of superfused hippocampal and cortical brain slices will be employed to explore some of the mechanisms responsible for changes in the expression and processing of ICE-family proteases during neuronal cell death, using specific protease inhibitors; and (5) transgenic mouse production and cre-lox-based organ-specific gene ablation in mice will be used to explore the functional significance of specific caspases or caspase- activators in neurons. Taken together, these studies will further the understanding of the mechanisms that control neuronal cell life and death during stroke.